Tolerability and Potency of Sequential and Repeated AAV Corneal Gene Therapy

摘要 粘多糖症I(MPS I)是一种常染色体隐性遗传性单基因遗传病，由MPS基因突变引起。 编码α-L艾杜糖醛酸酶的基因，是一种普遍存在的分解大糖分子的酶 称为糖胺多聚糖(GAG)。在没有IDUA的情况下，细胞内和细胞外的GAG积累会导致 在扩大的细胞/器官中导致一种多因素和潜在致命的疾病，这取决于 突变和相关严重程度[1，2]。MPS I在全球大约有3,000-8,000名患者， 症状包括70%的MPS I患者出现角膜混浊，这是导致 失明。目前，还没有治疗方法来解决MPS I角膜混浊和穿透性角膜移植 在这一高危人群中，由于移植排斥反应(&gt；70%)，不是目前的护理标准。至 针对这种致盲疾病，在过去的5年里，我们优化了腺相关病毒(AAV)基因 向角膜输送，包括RainBIO-1(RBIO-1)的设计和验证，这是一种优化的IDUA 适用于AAV基因治疗的cdna基因盒。在几份报告中，RBIO-1已经证明了 能够恢复MPS I患者细胞的IDUA活性，将WT人角膜中的IDUA活性提高10倍，以及 最值得注意的是，在犬单一角膜模型中预防和逆转MPS I角膜混浊 间质内注射。RBIO-1在WT兔角膜中的严格安全性研究(n=20)，证实完全 耐受性，即使在剂量比最低有效剂量高10倍的情况下，以及严格的媒介基因组控制 对注射后的角膜和视网膜进行观察。这些乐观的安全性和有效性数据已经定位 RBIO-1可能是治疗任何前部眼病的一流AAV疗法。关于一项临床方案 对于RBIO-1，最初在单个角膜上给药是评估MPS I安全性和有效性的最谨慎的方法 病人。如果RBIO-1缓解MPS I角膜堆积症，与从MPS I犬身上获得的结果相似， 对于双侧视力来说，需要对侧角膜进行顺序给药。这在临床上是这样的 将AAV基因顺序输送到角膜的相关问题将通过执行 目标1中提出的使用兔模型的实验。虽然，我们已经报告了强劲的IDUA产量和 在MPS I犬(n=10)上近1年的治疗成功和在兔身上6个月的治疗，AAV的持久性 转基因在角膜中的表达仍不清楚，也许将RBIO-1重新移植到单个角膜上可能会 是长期持久的疗效所必需的。目标2中的实验将确定AAV载体的范围 对先前给药的兔角膜重新给药后的转导。这里的工作将使用 RBIO-1用于确定连续注射AAV载体并在角膜重复给药的可行性 从而为治疗MPS I角膜混浊的I期方案设计提供关键数据。更广泛地说， 这里获得的关键数据的相关性从MPS I扩展到所有角膜，也许还有眼表， 也可能受益于AAV基因治疗方法的疾病。

Abstract Mucopolysaccharidosis I (MPS I) is an autosomal recessive monogenetic disorder caused by mutations in the gene encoding alpha-L-iduronidase (IDUA), a ubiquitous enzyme that breaks down large sugar molecules called glycosaminoglycans (GAGs). In the absence of IDUA, intra- and extra-cellular GAG accumulation results in enlarged cells/organs resulting in a multifactorial and potentially lethal disease depending upon the nature of the mutation and the correlative severity [1, 2]. MPS I afflicts approximately 3,000-8,000 patients worldwide, and symptoms include clouding of the cornea occurring in 70% of MPS I patients which is the leading cause of blindness. Currently, no therapeutics exist to address MPS I corneal clouding and penetrating keratoplasty is not a current standard of care due to transplant rejection (>70%) in this high-risk patient population. To address this blinding disorder, over the past 5 years we have optimized adeno-associated virus (AAV) gene delivery to the cornea, including the engineering and validation of RainBIO-1 (RBIO-1), an optimized IDUA cDNA genetic cassette amenable to AAV gene therapy. In several reports, RBIO-1 has demonstrated the ability to restore IDUA activity to MPS I patient cells, elevate IDUA activity in WT human corneas >10-fold, and most remarkably, prevent and reverse MPS I corneal opacity in a canine model following a single corneal intrastromal injection. Rigorous safety studies of RBIO-1 in WT rabbit corneas (n=20), demonstrate complete tolerability, even at doses 10-fold higher than the lowest effective dose, and strict vector genome containment to the injected cornea and retina was observed. These optimistic safety and efficacy data have positioned RBIO-1 as the likely first-in-class AAV therapeutic for any anterior ocular disease. Regarding a clinical protocol for RBIO-1, administration to a single cornea, initially, is most prudent to evaluate safety and efficacy in MPS I patients. If RBIO-1 alleviates MPS I corneal storage disease similar to results obtained from MPS I canines, sequential administration to the contralateral cornea would be desired for bilateral vision. This clinically relevant question of sequential AAV gene delivery to the cornea will be answered by the execution of the experiments proposed in Aim 1 using a rabbit model. Although, we have reported strong IDUA production and therapeutic success out nearly 1 year in MPS I canines (n=10) and 6 months in rabbits, the durability of AAV transgene expression in the cornea remains unknown, and perhaps redosing of RBIO-1 to a single cornea may be required for long-term sustained efficacy. The experiments in Aim 2 will determine the extent of AAV vector transduction following re-administration to a previously dosed rabbit cornea. The work herein will employ RBIO-1 to determine the feasibility of sequential AAV vector injections and repeat administration in the cornea and thereby provide critical data for Phase I protocol design to treat MPS I corneal clouding. More broadly, the relevance of the critical data obtained herein extends beyond MPS I to all corneal, and perhaps ocular surface, disorders which also may benefit from an AAV gene therapy approach.